The present invention relates to a novel sperm immotility protein isolated from an earthworm, Pheretima posthuma, belonging to the phylum, Annelida useful as an anti fertility agent. The invention also provides a method for the isolation of an active factor in the Coelmic Fluid (CF) of commonly available mature specimens of the earthworm, Pheretima posthuma belonging to the phylum, Annelida useful as an anti fertility agent.
Control of population explosion is a highly significant program related to the progress of any developing countries like India and China. There are some contraceptive devices and agents in the market, which are no doubt helpful in regulating the fertility and therefore population control but most of them pose problems either in relation to the total success or toxic side effects as in the case of the contraceptive pills which contains steroids. Hence, we really need a very successful device or product that will not only assure a total success but also would not produce harmful side effects.
Hence, attempts from various countries are directed to avail such a product and till date a total success has not been achieved. There is an interesting story in Japan. An ex-Emeritus Professor of Tokyo University with a group of investigators started a research program on the availability of a contraceptive agent from the coelomic fluid of Eisenia foetida. Addition of a factor from the coelomic fluid of Japanese earthworm, Eisenia foetida immediately stopped sperm motility of a number of vertebrates including human beings. The group was very excited and went on purifying the factor, which is a protein and found it to be a monomeric protein of 41 kDa. Later, when they even cloned the gene of this protein they suddenly discovered the high toxicity of this protein (Sekizawa et al., 1996, Biomed. Res., 17(3), 197-203; Sekizawa et al., 1997, Gene, 191, 97-102; Yamaji et al., 1998, J. Biol. Chem., 273(9), 5300-5306; Kobayashi et al., 2000, J. Expl. Zool., 286, 538-549). Injection of this protein into the blood immediately killed rats and mice. Addition of this protein into RBC preparation lysed the cells instantaneously. This incident spoiled their objective but they could observe another important aspect of this protein i.e. its specificity to bind sphingomyelin, a lipid invariably occurring in the cell membrane of majority of the vertebrates. It is due to this binding that the cell is lysed. However, they found a highly specific sphingomyelin binding protein, which is an exceptionally important protein as such proteins are still not available. It is named as lysenin and is marketed at high price due to its sphingomyelin binding property. They also published a number of papers on lysenin.
Clearly, their major objective was a failure i.e. its use as an agent for antifertility where commercial value would have been more.
The main object of this invention is to identify a factor from the Coelomic Fluid (CF) of an earthworm, which causes instant immotility of sperms and can be used as an anti-fertility agent.
Another object of the invention is to project the factor as a totally non-toxic compound as compared to the other contraceptive agents available in the market day, which manifest severe side effects.
The applicants initiated work with an Indian earthworm Pheretima posthuma, extracted the coelomic fluid and tested them on sperm motility and found that it also immediately stopped sperm movement, as effectively as lysenin. But there is a conspicuous difference. The extracted material from Indian earthworm did not produce any mortality of rats, mice or rabbits. A thorough examination on this toxicity showed no harmful effect on liver function or the function of other tissues. Moreover, its addition to RBC preparation did not produce any lysis of the cells as has been observed with lysenin. Hence, the extracted material has the property for causing instantaneous immotility of sperm without adverse side effects. This is a genuine advantage and could be profitably utilized for fertility control. This factor is also a protein. The applicants also have purified it through molecular exclusion chromatography (SG-100) followed by HPLC anionic chromatography. Hence, it is a new product from an Indian earthworm source and requires protection.